Figure 3.

ERdj4 Opposes IRE1 Dimerization

(A) Crystal structure of human IRE1^LD (PDB: 2HZ6) highlighting Q105 (black) at the dimer interface.

(B) Reducing Phos-tag SDS-PAGE of endogenous IRE1α recovered from WT or IRE1^Q105C cells treated in the indicated manner. Fraction of active (phosphorylated) IRE1-P from this representative immuno blot is noted.

(C) Representative immunoblot of endogenous IRE1α and PERK recovered from the indicated cell lines by immunoprecipitation of IRE1α or PERK and resolved by reducing and non-reducing SDS-PAGE. ER stress was induced by thapsigargin (Tg) or DTT.

(D) Schema of IRE1^{LD Q105C}-GST with the Q105C-Q105C disulfide indicated.

(E) Representative immunoblot of IRE1^{LD Q105C}-GST and BiP recovered from ΔERdj4 cells transfected with indicated constructs and resolved by non-reducing SDS-PAGE.

(F) Ratio of disulfide-bound IRE1^LD Q105C-GST dimer to free thiol in indicated samples. Quantified in six independent experiments as shown in (D) above (mean ± SD, n = 6, ^∗∗∗∗p < 0.0001, unpaired Student’s t test with Welch’s correction).

See also Figure S2.